| In the past years, evidence has accumulated that effective immunity is elicited by a delicate balance of intricate interactions between multiple immune effector cells. Dendritic cells (DCs) and natural killer (NK) cells represent two types of specialized cell of the immune system. Since 1999, more attention has focused on interactions between DCs and NK cells. In vitro and in vivo studies have demonstrated various effects of DC-NK interactions, including activation and cytokine production, maturation of DCs and NK-cell lysis of immature DCs in peripheral tissues or certain mature DCs in lymphoid nodes. It has been reported that NK-cell recognition and lysis of host DCs might also provide new methods to prevent GvHD in allogeneic transplants. So, cognate interactions between NK cells and DCs were useful for defence against tumor, infection, or improving the outcome of allogeneic transplants or vaccine protocol. However, DCs or NK cells can be categorized into different subpopulations, which have distinct regulatory functions in the immune response. Up to now, the relationships between these subsets have not been discussed yet.Our previous studies have identified an undescribed regulatory DC subset (diffDCs), which were developed from differentiation of mDCs educated by splenic stromal cells.diffDCs secret high levels of IL-10 and NO, and inhibit proliferation of activated CD4+ T cells, suggesting its negative feedback control on the immune response in the late phage. These past findings pave the way for the new opinion that mDCs are not terminal differentiation, and also identify an important mechanism by which the microenvironment regulates the immune response. Based on these past work, in the present study, we firstly demonstrated that IL-10 secretion of diffDCs by LPS stimulation was dependent on activation of MAPKs. Importantly, we revealed that diffDCs can activate spleen-derived resting NK cells at indicated DC/NK ratios. Activated NK cells secreted IFN-γ and acquired cellular cytotoxicity on target cells. These phenomena were more obvious in diffDCs stimulated by LPS. Furthermore, we demonstrated that the activating effects of diffDCs on splenic NK cells were partly mediated by diffDC-derived IL-10, one cytokine initially characterized as a potent suppressor of the cell-mediated immune response. Interestingly, we also observed that diffDC-activated NK cells could kill some freshly purified diffDCs in turn, although the mechanisms involved need more investigation. Together, our data suggest that diffDC-NK-cell crosstalk in spleen might play an important regulatory role both in innate and adaptive responses.Part I: The impact of diffDCs on splenic NK cells and the possible mechanisms1. The relevant signal pathways involved in IL-10 secretion of diffDCsHigh level of IL-10 is one of main characters of diffDCs, with more pronounced afterLPS stimulation. So, we firstly focused our interests on the possible mechanisms involved in IL-10 secretion of diffDCs. maDCs or diffDCs were purified and then stimulated with or without 500 ng/ml LPS for 24 h, then were harvested for FACS analysis. IL-10 and IL-12p70 were also determined in the supematants. In some cases, purified maDCs or diffDCs were stimulated with 500ng/ml LPS for different time (0,10,20,40,60min), and then were collected for Western blot analysis. Interestingly, we found, diffDCs secreted significantly more IL-10 but no obvious changes were observed in its phenotype. ERK, JNK, p38 were phosphated when diffDCs were stimulated by LPS. Phosphorylation of ERK was more obvious, even occurred in diffDCs without LPS stimulation. In addition, translocation of NF-kB to the nucleus was found in diffDCs with or without LPS stimulation. IL-10 secretion of diffDCs with LPS stimulation was significantly reduced if ERK, JNK or p38 was blocked priorly using specific inhibitors, respectively. However, no decreasing of IL-10 production was found in diffDCs if NF-kB was blocked priorly. These results indicated that LPS seems to have no effects on phenotype of diffDCs, but could...
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